Current research in this laboratory focuses on the way in which membrane glycoproteins are expressed at their site of synthesis in the rough endoplasmic reticulum and what particular properties of the protein sequence and carbohydrate microstructure are responsible for the sorting of the membrane glycoproteins to their final membrane compartment. We are studying particular membrane glycoproteins in the rough endoplasmic reticulum by using membrane maturing viruses as a probe. Rotavirus SA11 matures by budding into the RER of infected cells. The membrane, initially acquired from the host cell (RER) membrane, is later lost giving rise to the double protein shelled virus typical of the Reoviridae. The structural glycoprotein VP7, also the neutralizing antigen, and the nonstructural glycoprotein, NCVP5, have been located to the RER by immunoelectronmicroscopy and by immunofluorescence. These integral membrane glycoproteins are not found in the Golgi apparatus or the plasma membrane using the cloned genes. We are performing in vitro site-specific mutagenesis in order to identify regions in the gene products which are important in the RER location of the molecules. Functional portions of the genes of glycoproteins from Semliki forest virus (SFV) and vesicular stomatitis virus (VSV), which bud from the plasma membrane, are being inserted in the rotavirus genes in order to induce the products to move from the RER and thereby demonstrate the molecular basis for such translocation. The oligosaccharide portion of the glycoproteins undergo a set of specific processing steps by enzymes located in the RER and the Golgi appartus. The form of the oligosaccharide therefore provides an indication of where the glycoprotein are located in the cell and what compartments they have already migrated through. In some cases, a particular form of N-linked oligosaccharide is necessary for the intracellular migration of membrane and secretory glycoproteins. We are using high field (500 MHz) spectoscopy in combination with conventional complex carbohydrate chemistry to elucidate the molecular structure of N-linked glycoprotein oligosaccharides. We plan to seek alpha 1,2-mannosidases which may be growth-dependent and also located in the rough endoplasmic reticulum. (A)